The Hedgehog (Hh) signaling molecule is a secreted autoproteolytic protein that activates the Hedgehog protein signaling pathway, which is a signaling pathway that plays a fundamental role in the morphogenesis of many tissues, in particular in the formation of the endoderm and of the embryonic axis, the development of the brain and hair follicles, and also in cell proliferation, and appears to be involved in tissue maintenance and repair in adults (Ingham et al., Genes Dev., 2001, 15, 3059-3087; Marti et al., Trends Neurosci., 2002, 25, 89-96; Weschler et al., Annu. Rev. Neurosci., 2001, 24, 385-428).
The Hedgehog protein and the associated transduction pathway, initially demonstrated in drosophila, are conserved in vertebrates and invertebrates. A single homolog of Hh is present in drosophila, while three homologs of Hh: Sonic (Shh), Indian (Ihh) and Desert (Dhh) are present in mammals. Among these three homologs, Shh has been the most widely studied owing to its extended expression profile during development. Shh participates in neural tube ventralization by specifying the early phenotype of several types of neurons along the ventral median line (spinal cord motoneurons, dopaminergic or cholinergic neurons) and by inducing the generation of oligodendrocyte precursors from the ventral spinal cord. Moreover, Shh induces survival of gabaergic and dopaminergic neurons, orientates the fate of serotoninergic precursors and prevents dopaminergic neuron death caused by the toxin MPP. Finally, it induces granular cell precursor proliferation in the early post-natal cerebelum. The other members of the Hedgehog family participate, for their part, respectively in the development of bone tissue (Ihh), the testicles and the peripheral nerves (Dhh). In addition, the results obtained with Shh are also applicable to Dhh and Ihh.
The regulatory role of the Hedgehog protein signaling pathway during embryonic development has been widely studied: Hh has been associated with maintenance and repair processes in normal tissue, and in the spatiotemporal regulation of proliferation and differentiation, thus allowing developing tissues to reach their correct size with the appropriate cell types and appropriate degrees of vascularization and innvervation. The essential role of the Hh signaling function is demonstrated by the dramatic consequences of defects in this signaling pathway in the human fetus, such as holoprosencephaly observed in Shh mutants.
More recently, the Shh pathway has been identified in the adult brain, where the amino-terminal active form of the molecule is expressed in many regions of the mature nervous system at a higher level than that encountered during the early post-natal period (Traiffort et al., Eur. J. Neurosci., 1999, 11, 3199-3214 et 2001, 14, 839-850). Although the roles of Shh in adults have not been completely elucidated, it first appeared, like other neutrotrophic molecules, to be a factor capable of promoting survival and maintenance of the phenotype of cells of the nervous system (Reilly et al., Mol. Cell. Neurosci., 2002, 19, 88-96; Charytoniuk et al., Eur. J. Neurosci., 2002, 16, 2351-2357). Under pathological conditions, such as a model for Parkinson's disease or a model for peripheral neuropathy, Shh is capable of preserving axonal projections of dopaminergic neurons in the striatum or of improving the time required for motor recovering subsequent to crushing of the sciatic nerve (Tsuboi et al., Exp. Neurol., 2002, 173, 95-104; Pepinski et al., J. Pharm. Sci., 2002, 91, 371-387).
Hh proteins are synthesized in the form of immature precursors of approximately 45 kDa which undergo intramolecular cleavage catalyzed by the C-terminal region of the precursor. This cleavage produces a 25 kDa C-terminal fragment with no known additional function and a 19 kDa active amino-terminal fragment (called HhNp for N-terminal processed domain) linked at its C-terminal end to a cholesterol molecule, sufficient for all the known signaling activities of Hedgehog proteins.
The Hedgehog protein signaling pathway comprises three main components; the Hh ligand, a transmembrane receptor circuit composed of the Patched (Ptc) negative regulator and the Smoothened (Smo) activator, and a cytoplasmic complex which regulates the transcriptional effectors.
The cellular response to the Hedgehog morphogen is controlled by the expression products of the Patched (Ptc) gene, which is a tumor suppressor gene, and of the Smoothened (Smo) protooncogene; however, the exact mechanism for Hedgehog pathway regulation has not been completely elucidated. In mammals, there are two Patched genes encoding respectively Ptc1 and Ptc2, glycoproteins with 12 transmembrane domains which are homologous to bacterial transporters. The product of the Smo gene which encodes a protein of the G protein-coupled receptor family has no known endogenous ligand. In the absence of Hedgehog proteins, Ptc appears to block the constitutive activity of Smo. The binding of Hedgehog to Ptc appears to lift this inhibition and to allow signal transduction by means of Smo. The mechanism for regulating the activity of Smo by Ptc, in mammals, could involve a molecule transported by Ptc and interacting with Smo (Taipale et al., Nature, 2002, 418, 892-896). The activation of Gli transcription factors is involved in the cascade of events resulting from the activity of Smo. The type I transmembrane protein, HIP (Hedgehog Interacting Protein), constitutes another receptor for Hedgehog molecules which it binds with an affinity comparable to that of Ptc; HIP has been proposed as a negative regulator of the pathway (Ingham et al., mentioned above; Ho et al., Curr. Opin. Neurobiol., 2002, 12, 57-63; Taipale et al., Nature, 2001, 411, 349-354). In addition, the products of the dispatched (Disp) gene, in particular DispA, appear to be involved in the release of Hedgehog proteins in the soluble form into the extracellular medium and the accumulation thereof in said medium (Ma et al., Cell, 2002, 111, 63-75).
Shh signaling pathway dysfunctions have been associated with numerous cancers, in particular following the characterization of Ptc as a tumor suppressor gene. Indeed, inactivating mutations of Ptc are associated with Gorlin syndrome or basal cell nevus syndrome, an autosomal dominant disease characterized by cranofacial and cerebral malformations, particularly by a high incidence of various tumors, more particularly basal cell carcinomas of the skin and medulloblastomas in the brain. Mice heterozygous for the Ptc gene develop cerebellar tumors, suggesting that a modification of the Shh pathway is responsible for these tumors (Goodrich et al., Science, 1997, 277, 1109-1113).
Mutations of human Ptc or Smo genes are also observed in primitive neuroectodermal tumors of the central nervous system, mainly medulloblastomas (30% of cases), but also in sporadic forms of basal cell carcinomas (respectively 40% to 20% of cases for Ptc and Smo). In addition, mutations in Shh (H133Y) are also associated with basal cell carcinomas. Smo mutations, which mainly involve two amino acids located in the seventh hydrophobic domain of the receptor (W535L and S533N), induce constitutive activation of the pathway which escapes the negative control of Ptc. In contrast, those of Ptc result in a reduction in the inhibition exerted thereby on Smo in the absence of Shh. In the two cases, activation of the Shh pathway results therefrom and leads to a powerful mitogenic activity demonstrated in cultures of precursors of granular cells of the developing cerebellum, and to blocking of the terminal step of differentiation of these neuroblasts (Traiffort et al., Eur. J; Neurosci., 1999, mentioned above; Charytoniuk et al., J. Physiol. Paris, 2002, 96, 9-16; Dahmane et al., Development, 1999, 126, 3089-3100; Wallace et al., Curr. Biol., 1999, 22, 103-114; Weshler-Reya et al., Neuron., 1999, 22, 103-114). Likewise, the expression of Smo carrying one of these mutations in transgenic mice results in the presence of basal cell carcinomas, thereby demonstrating the direct involvement of Smo in the development of these tumors (Xie et al., Nature, 1998, 391, 90-92).
Apart from basal cell carcinomas and medulloblastomas, other types of tumors have been associated with a defect in the Hedgehog signaling pathway; the location of these tumors is closely correlated with the sites of expression of the components of the pathway during embryonic development. By way of nonlimiting example, mention may be made of: breast cancers and meningiomas associated with Ptc mutations, glioblastomas associated with Gli mutations, gastrointestinal cancers, in particular primary cancers of the stomach, prostate cancers, ovarian fibromas and dermoids, rhabdomyosarcomas, small cell lung cancers, an oral squamous cell carcinomas. Recently, Shh has been associated with psoriasis.
Because of the essential role of the Hedgehog protein signaling pathway in numerous physiological processes and consequently of the significance of the pathological conditions linked to the dysfunction thereof, the components of this pathway, such as the Smoothened and Patched (Patched 1 and Patched 2) proteins, the Dispatched (Dispatched 1 and Dispatched 2) proteins or else the HIP protein, represent targets for developing novel molecules capable of modulating (activating or inhibiting) this pathway and thus upregulating or downregulating the development [proliferation, differentiation, migration, survival (apoptosis)] and/or the activity of differentiated cells and stem cells, in vitro and/or in vivo, in embryos or in adults.
Such molecules are of use in the treatment of tumors associated with hyperactivation of the Hedgehog pathway: nervous tissue tumors (medulloblastomas, primative neuroectodermal tumors, glioblastomas, meningiomas and oligodendrogliomas), skin tumors (basal cell carcinomas, trichoepitheliomas), muscle and bone tissue tumors (rhabdomyosarcomas, osteosarcomas) and tumors of other tissues (kidney, bladder).
Such molecules are also of use in the treatment of neurodegenerative pathological conditions requiring blocking of the Hedgehog pathway (Parkinson's disease, Huntington's chorea, Alzheimer's disease, multiple sclerosis, motoneuron disease), and diseases in which blocking of the Hedgehog signaling pathway could be beneficial, such as diabetes.
Such molecules are also of use in the medical or surgical treatment (plastic or reconstructive surgery, tissue or organ transplantation) of numerous acute, subacute or chronic, genetic or acquired pathological conditions—involving a tissue dysfunction linked to disregulation of the Hedgehog pathway—, for inducing the formation, regeneration, repair and/or increase in activity of tissues such as, in a nonlimiting manner: nerve tissue [central nervous system (brain) and peripheral nervous system (sensory neurons, motor neurons, sympathetic neurons)], bone, cartilage, testicles, liver, spleen, intestine, pancreas, kidneys, smooth and skeletal muscles, heart, lungs, skin and hair system, mucous membranes, blood cells and cells of the immune system. By way of nonlimiting example of these pathological conditions, mention may in particular be made of neuropathies and associated neuromuscular diseases, diabetes, alopecia, burns, ulcers (skin and mucous membranes) and problems with spermatogenesis.
Various molecules, capable of modulating the activity of the Hedgehog pathway, have been identified:                Hedgehog proteins and derived polypeptides (fragments, variants, etc.), in particular Hedgehog protein agonists and antagonists (PCT International Application WO 01/98344 in the name of Biogen); owing to their size, these proteins and the derived polypeptides cannot cross the blood-brain barrier and cannot therefore be administered systemically, in particular for the treatment of brain tumors linked to hyperactivation of the Hedgehog protein signaling pathway. In addition, such molecules are not very stable and are difficult to produce and to purify;        heterocyclic organic molecules (PCT International Application WO 01/74344 in the name of Curis and Chen et al., PNAS, 2002, 99, 14071-14076);        nitrogenous heterocyclic molecules (PCT International Applications WO 01/19800, WO 01/26644 and WO 02/30421 in the name of Curis and Kamenetsky et al., J. Biol., 2002, 1, 1-19); and        plant steroids derived from Veratrum spp (jervine, cyclopamine and cycloposine) and from Solanum spp. (solanidine), substituted in position 16, 17 or 18 with an amine or an amine derivative, and cholesterol: U.S. Pat. No. 6,432,970 and PCT International Applications WO 99/52534 and WO 01/27135 in the name of Johns Hopkins University School of Medicine; U.S. Pat. No. 6,291,516; PCT International Application WO 00/41545 in the name of Ontogeny Inc.; PCT International Application WO 02/30462 in the name of Curis; Talpale et al., Nature, 2000, 406, 1005-1009; Berman et al., Science, 2002, 297, 1559-1561). However, cyclopamine is a teratogenic agent responsible for holoprosencephaly and cyclopia in the embryo in mammals, and it has also been demonstrated that cyclopamine concentrations above 10 μM prove to be cytotoxic for cells (Borzillo et al., Curr. Top Med. Chem., 2005, 5(2), 147-57). With regard to the other compounds derived from plant steroids, the absence of toxicity thereof in mammals has not yet been demonstrated;        mifepristone (17β-hydroxy-11β-(4-dimethylaminophenyl)-17α-(prop-1-ynyl)estra-4,9-dien 3-one), also called RU-486 or RU-38486 (French patent FR 2 850 022 in the name of CNRS), for which an inhibitory activity of the activity of the Hedgehog protein signaling pathway has been demonstrated;        urea or thiourea derivatives which are antagonists of the Hedgehog protein signaling pathway have also been described in application US 2005/0085519 A1.        
The molecules SANT74 and SANT75 which have a structure analogous to that of SAG, a synthetic activator compound of chlorobenzothiophene type (CAS No.: 364590-63-6) corresponding to the following formula:
are also known to be stable inhibitors which make it possible to effectively control the conformation of the Smo activator (Yang et al., The Journal of Biological Chemistry, published on Apr. 14, 2009).
Other compounds inhibiting the Hedgehog signaling pathway have also been described recently: pyridyl-based inhibitors (PCT International Application WO 2006/028958 in the name of Genentech Inc. and Curis Inc.) and bisamide-based inhibitors (PCT International Application WO 2007/059157 in the name of Genentech Inc. and Curis Inc.).
Other molecules which act in particular on the transcription factors of the Gli family have also been described (Mahindroo et al., J. Med. Chem. 2009, 52, 3829-3845).
It follows from the aforementioned that there is currently no molecule which makes it possible to modulate the activity of the Hedgehog protein signaling pathway and for which an absence of toxicity has been established by means of clinical trials in humans.